DE60105753T2 - Process for the preparation of a gluten-free peptide preparation and preparation thereby obtained - Google Patents

Abstract

The invention relates to a method for producing a glutamine-rich gluten-free peptide preparation from gluten protein, comprising the steps of enzymatically hydrolysing wheat gluten using one or more proteases to obtain a hydrolysate; acidifying the hydrolysate to a pH between 4 and 5; and filtering the hydrolysate to obtain the glutamine-rich gluten-free peptide preparation as the filtrate. The protease is preferably a neutral or basic protease and the optimum pH is from 4.5 to 4.7.

Description

The The present invention relates to a method of manufacture a peptide preparation that is both glutamine-rich and gluten-free is, and on the resulting preparation. The invention relates Furthermore on the use of the preparation in different products and on the products containing the preparation.

gluten is a combination of proteins that are different in the endosperm Cereals such as wheat, barley and rye, oats and others Wheat variants containing gluten, such as triticale, spelled and Kamut, occurs. In wheat, gluten makes 90% of the protein and almost 15% of the total weight of a grain. It is therefore important Source for Protein.

gluten is the cause of a genetic disorder, as a celiac disease or gluten intolerance is known. Symptoms of celiac disease can be of classic features such as diarrhea, weight loss and malnutrition latent symptoms, such as individual nutrient deficiencies. The disease usually affects people European Appearance and occurs less frequently in black and Asian populations on. The victims suffer damage to the villi (shortening and Villi flattening) in the lamina propria and crypt areas of their Guts, if they have specific cereal antigens (toxic amino acid sequences) eat in wheat, rye and barley, oats and other gluten containing wheat variants, such as Triticale, Spelled and Kamut, occurrence. The gluten found in rice and corn does not cause any Intolerance.

For persons with celiac disease the toxic part of the gluten molecule is the prolamin domain: gliadin in wheat, secalin in rye and horedin in barley. By following a gluten free diet can People recover from the symptoms of the disease, but they can not be cured. A reintroduction of gluten into the diet will be renewed lead to symptoms.

Glutamine is an amino acid that is abundant in gluten. Although not an essential amino acid, it is nonetheless desirable for certain individuals, particularly those recovering from surgery, suffering from gastrointestinal dysfunction, immune function deficiencies, metabolic stress, shock, or endurance sports. Such individuals would benefit from supplementation with this amino acid, for example, by taking a peptide preparation rich in glutamine. EP 0 672 352 discloses a process for the preparation of glutamine-rich peptide mixtures comprising hydrolysis of a glutamine-rich protein (eg wheat protein) by an endopeptidase (eg papain), lowering the pH to 4.0, heating to inactivate the enzyme, and a Centrifugation to partially remove the undissolved material.

gluten is a very cost effective Source for such glutamine-rich peptide preparations. The known preparations are not for celiac disease patients suitable because they still contain the toxic parts of gliadin.

Therefore it is the object of the present invention to provide a peptide preparation which is rich in bound glutamine, but at the same time is gluten free.

• a) enzymatisches Hydrolysieren von Gluten unter Verwendung einer oder mehrerer Proteasen, um ein Hydrolysat zu erhalten;
• b) Ansäuern des Hydrolysats auf einen pH-Wert zwischen 4 und 5; und
• c) Filtrieren des Hydrolysats, um die glutaminreiche, glutenfreie Peptidzubereitung als das Filtrat zu erhalten.

Such a peptide preparation can be obtained by a method comprising the steps of:

• a) enzymatically hydrolyzing gluten using one or more proteases to obtain a hydrolyzate;
• b) acidifying the hydrolyzate to a pH between 4 and 5; and
• c) filtering the hydrolyzate to obtain the glutamine-rich, gluten-free peptide preparation as the filtrate.

Of the Term "gluten-free" should indicate that the product, when tested in an ELISA based on anti-Ω-gliadin antibodies, a value of <200 ppm results. A suitable ELISA to test gluten-free is in "Association of Official Analytical Chemists' (AOAC's) Official Methods of Analysis, 15th Edition, 2nd supplement (1991) ".

It will be appreciated that the proteases to be used may be selected from a wide range of proteases known to those skilled in the art, provided that the hydrolysis carried out with such a protease yields a formulation containing less than 200 ppm in the prior art gives the ELISA described above. The proteases include acidic, basic and neutral proteases derived from bacterial, fungal, animal or plant sources. It has been found that basic or neutral proteases which are active at a pH above 6 are particularly well suited. Examples of such proteases are Proleather N (Amano), Neutrase (NOVO), PROMOD 192P (Biocatalysts), Alcalase 2.4L (NOVO), Protease S (Amano), Peptidase A (Amano), Peptidase R (Amano). Of these, the following proteases are preferred: Proleather N (Amano) and Alcalase 2.4L (NOVO).

The Protein fragments that cause hypersensitivity in celiac disease patients are surprisingly removed when the hydrolyzate is acidified and then filtered becomes. It is believed that these fragments are precipitated and behind the filter back stay. The pH to which the hydrolyzate is acidified must be between 4 and 5, and preferably between 4.1 and 4.9, more preferably between 4.3 and 4.8, most preferably between 4.5 and 4.7 and is optimally 4.6.

The Hydrolysis is an essential step in the process according to the invention, since without hydrolysis, the toxic fragments are not removed can.

Peptide preparations that by the method according to the invention available are and consist of peptides that have no gluten hypersensitivity symptoms in celiac disease patients Calling forth are another aspect of this invention. Such preparations are suitable as a food additive or food to a person extra To supply glutamine. The preparation is therefore used in sports and in the clinic and can for the enteral nutrition and pet foods are used.

The Peptide preparation of the invention may be used in other products be taken by persons who need a supplement or can be administered to them. Special embodiments for such Products are glutamine peptide tablets, which are the usual Carrier, thinner and excipients for tablets and a peptide preparation of the invention as a glutamine peptide source include, glutamine peptide liquid drinks which the usual ingredients for beverages and a peptide preparation of the invention as a glutamine peptide source and glutamine-peptide-neutral foods, which are the usual ones Carrier, thinner and excipients for enteral nutrition and a peptide preparation of the invention as a glutamine peptide source includes.

Even though the invention very generally gluten of all grains, the celiac Disease can cause, applicable is preferred to use wheat because of its high glutamine content.

The The present invention will be further explained in the following examples. the only for explanation and in no way the scope of the invention restrict should.

Examples

example 1

Producing a glutamine-rich, gluten-free peptide preparation

A Series of experiments was carried out to the critical process parameters to illustrate.

A Series of peptide hydrolysates were prepared by heating deionized water to a temperature of 63 ° C ± 1 ° C. To this water becomes one Mixture of 45% liquid Potassium hydroxide, 50% liquid Sodium hydroxide, hydrated calcium hydroxide in a ratio of 1: 0.78: 0.70 respectively, to obtain a pH value for the suitable protease to use.

Vitales Wheat gluten ("VWG", Cargill B.V., Bergen op Zoom, The Netherlands) is added to this solution to give a 12% solids mixture from soluble to make gluten.

hydrolysis with a desired Protease is carried out as indicated in the description of the individual experiments below. The hydrolysis reaction is for 3 hours at a temperature suitable for the protease used is, usually 60 ° C ± 2 ° C.

After hydrolysis, acid, especially sulfuric acid, is added with stirring to reach the desired pH (see description of the experiments). The reaction is terminated by HTST (high temperature short time) heating at 116 ° C ± 2 ° C. The solution is then heated to 66 ° C ± 2 ° C cooled and filtered using diatomaceous earth (Eagle-Picher Minerals Inc., Reno, NV) at 40% filter aid. The solution is circulated through the filter press for a minimum of 3 minutes.

Of the pH of the filtrate is adjusted to 6.4 to 6.8 by means of an alkaline solution set. After evaporation of the liquid and drying obtained a powdered peptide preparation of the invention.

Around To test whether the product is gluten-free, an ELISA according to ADAC 991.19 (Official Methods of Analysis (1990), 15th Edition, 2nd Supplement (1991)).

Of the bound glutamine content was determined according to P.E. Wilcox, "Determination of Amide Residues by Chemical Methods ", Methods of Enzymology 11, 63-76 (1967) certainly.

One Measure of the degree of hydrolysis of protein is the AN / TN ratio. AN is the amine nitrogen level produced by the mold titration method can be determined or according to J. Adler-Nissen, "Enzymatic hydrolysis of food proteins ". Elsevier Applied Science Publishers, 1986. TN is the total amine nitrogen content which according to the Kjeldahl nitrogen determination method is determined. The bigger that relationship AN / TN is, the bigger the degree of hydrolysis of the protein preparation.

experiments

Experiment 1

wheat gluten is dispersed in water. The pH is raised with sulfuric acid 4.6 adjusted and the solution filtered.

Experiment 2

wheat gluten is dispersed in water. The pH is raised with sulfuric acid 3.2 to 3.4 set. The gluten is made using acid Protease II (Amano) digested. The enzyme is heat inactivated and the solution filtered.

Experiment 3

wheat gluten is in caustic water dispersed. The gluten is made using alkaline and neutral Proteases Alcalase 2.4L (NOVO) and Proleather N (Amano) and Amylases Digested BAN 240L (NOVO). After inactivation of the enzymes, the pH with sulfuric acid adjusted to about neutral, and the solution is filtered.

Experiment 4

wheat gluten is in caustic water dispersed. The gluten is made using alkaline and neutral Proteases Alcalase 2.4L (NOVO) and Proleather N (Amano) digested. The pH is adjusted with sulfuric acid set to 3.8 to 4.1. After heat inactivation of the enzymes becomes the solution filtered. The pH is then adjusted to neutral with caustic.

Experiment 5

wheat gluten is in caustic water dispersed. The gluten is made using alkaline and neutral Proteases Alcalase 2.4L (NOVO) and Proleather N (Amano) digested. The pH is adjusted with sulfuric acid set to 6.5. After heat inactivation of the enzymes, the solution filtered. The pH is measured using caustic neutral.

Experiment 6

wheat gluten is in caustic water dispersed. The gluten is made using alkaline and neutral Proteases Alcalase 2.4L (NOVO) and Proleather N (Amano) digested. The pH is adjusted with sulfuric acid set to 4.3. After heat inactivation of the enzymes and filtration the pH is adjusted to neutral using caustic.

Experiment 7

wheat gluten is in caustic water dispersed. The gluten is made using alkaline and neutral Proteases Alcalase 2.4L (NOVO) and Proleather N (Amano) digested. Subsequently, the pH is adjusted to 4.5 with sulfuric acid. To Heat inactivation of the enzymes and filtration will lower the pH Use of etchant neutral again.

Experiment 8

wheat gluten is in caustic water dispersed. The gluten is made using alkaline and neutral Proteases Alcalase 2.4L (NOVO) and Proleather N (Amano) digested. The pH is adjusted with sulfuric acid set to 4.6. After heat inactivation of the enzymes, the solution filtered. Then it becomes pH using caustic neutral.

Experiment 9

wheat gluten is in caustic water dispersed. The gluten is made using alkaline and neutral Proteases Alcalase 2.4L (NOVO) and Proleather N (Amano) digested. The pH is adjusted with sulfuric acid set to 4.8. After heat inactivation of the enzymes and filtration the pH is adjusted to neutral using caustic.

table 1 shows the results of the experiments. From the example above it becomes clear that both the hydrolysis of gluten and the filtration at an acidic pH are essential for making the product gluten-free is.

Table 1

Example 2

Application of gluten-free, glutamine-rich peptide preparation of the invention

in the Following are three examples of applications of the preparation given the invention.

1. glutamine peptide tablets

• (1) Enzymatisch hydrolysiertes Weizenprotein (granular) (Zubereitung gemäß der Erfindung)
• (2) Pharmacel 102
• (3) CAB-O-SIL M-5

Rezeptur: Enzymatisch hydrolysiertes Weizenprotein (1) 91,1 % Mikrokristalline Zellulose (2) 5,0 % Dicalciumphosphat 2,0 % Siliziumdioxid (3) 0,9 % Stearinsäure 0,5 % Magnesiumstearat 0,5 % Gesamtmenge 100 % ingredients:

• (1) Enzymatically hydrolyzed wheat protein (granular) (preparation according to the invention)
• (2) Pharmacel 102
• (3) CAB-O-SIL M-5

recipe: Enzymatically hydrolyzed wheat protein (1) 91.1% Microcrystalline cellulose (2) 5.0% dicalcium 2.0% Silicon dioxide (3) 0.9% stearic acid 0.5% magnesium stearate 0.5% total quantity 100%

Production method:

The powders are premixed (retention of the magnesium stearate until the last minutes of mixing). The tablets are made by direct compression. Properties of the tablets: Glutamine peptide per tablet 170 mg tablet weight 758 mg Tablet length (oblong) 19.04 mm compression pressure 13.3 kN hardness 140 N

2. glutamine peptide liquid beverage

• (1) Enzymatisch hydrolysiertes Weizenprotein (Zubereitung der Erfindung)
• (2) Enzymatisch hydrolysiertes Molkeprotein (WE80BG, DMV International)
• (3) Grapefruitaroma Tastemaker 946068

Rezeptes: Wasser (QS auf 1 Liter) 920,00 g Enzymatisch hydrolysiertes Weizengluten (1) 13,21 g Enzymatisch hydrolysierte Molke (2) 13,04 g Sukrose 26,60 g Glukose 15,00 g Fruktose 5,00 g Glukosepolymere (Maltodextrin DE18) 10,00 g Apfelsäure 3,33 g Zitronensäure 0,67 g Natriumcitrat 1,00 g Grapefruitaroma (3) 0,60 g Aspartam 0,10 g Kaliumacesulfam 0,10 g 1.000,00 ml ingredients:

• (1) Enzymatically Hydrolyzed Wheat Protein (Preparation of the Invention)
• (2) Enzymatically Hydrolyzed Whey Protein (WE80BG, DMV International)
• (3) Grapefruit flavor Tastemaker 946068

recipe: Water (QS to 1 liter) 920.00 g Enzymatically hydrolyzed wheat gluten (1) 13.21 g Enzymatically hydrolyzed whey (2) 13.04 g sucrose 26.60 g glucose 15.00 g fructose 5.00 g Glucose polymers (maltodextrin DE18) 10.00 g malic acid 3.33 g citric acid 0.67 g sodium citrate 1.00 g Grapefruit flavor (3) 0.60 g aspartame 0.10 g acesulfame potassium 0.10 g 1,000.00 ml

Production method:

All ingredients are added to the water and mixed well. The acids are added last to obtain a pH of 3.9. The liquid is bottled, heat treated for 1 minute at 85 ° C and cooled. Nutrient information (per 100ml): protein 2.09 g Glutaminpeptid 0.26 g carbohydrates 6.0 g

3. Clinical enteral nutrition prototype with glutamine peptide and whey peptides

• (1) Enzymatisch hydrolysiertes Weizenprotein (Zubereitung der Erfindung)
• (2) Enzymatisch hydrolysiertes Molkeprotein (WE80BG, DMV International)

Rezeptur: Wasser (QS auf 1 Liter) 720,00 g Enzymatisch hydrolysiertes Weizengluten (1) 40,00 g Enzymatisch hydrolysierte Molke (2) 35,60 g Speisestärke, modifiziert 84,00 g Maltodextrin 59,00 g Sojaöl 30,00 g MCT-Öl 10,00 g Kaliumcitrat 2,20 g Natriumcitrat 1,60 g Magnesiumchlorid 3,20 g Calciumphosphat 2,80 g Kaliumphosphat 2,00 g Natriumphosphat 1,00 g Carrageenan 0,50 g 1.000,00 ml ingredients:

• (1) Enzymatically Hydrolyzed Wheat Protein (Preparation of the Invention)
• (2) Enzymatically Hydrolyzed Whey Protein (WE80BG, DMV International)

recipe: Water (QS to 1 liter) 720.00 g Enzymatically hydrolyzed wheat gluten (1) 40.00 g Enzymatically hydrolyzed whey (2) 35.60 g Cornstarch, modified 84.00 g maltodextrin 59.00 g soybean oil 30.00 g MCT oil 10.00 g potassium citrate 2.20 g sodium citrate 1.60 g magnesium chloride 3.20 g calcium phosphate 2.80 g potassium phosphate 2,00 g sodium phosphate 1.00 g carrageenan 0.50 g 1,000.00 ml

Production method:

The Minerals are dissolved in water with constant stirring. The Premixed carbohydrates are added to the mixture. The Mixture is at 70 ° C heated and for 10 minutes with constant stirring held. The protein is added to the mixture, which is then submerged continuous stirring to 70 ° C heated becomes.

The oil is added to the mixture, which is then mixed well. The mixture is then double homogenized at 4000 psig (276 bar). The pH is adjusted to the appropriate value. The solids content is adjusted to a suitable value. The product is sterilized and the heat treatment is autoclaved at 121 ° C for 10 minutes. Nutrient information (per 100ml): protein 6.0 g Glutaminpeptid 1.0 g carbohydrates 13.8 g fat 4.0 g

Claims (16)

Process for producing a glutamine-rich, gluten-free peptide preparation of gluten protein, which is the following Steps includes: a) enzymatic hydrolysis of gluten using one or more proteases to obtain a hydrolyzate to obtain; b) Acidification the hydrolyzate to a pH between 4 and 5; and c) Filter the hydrolyzate to the glutamine-rich, gluten-free peptide preparation as the filtrate to get. Method according to claim 1, wherein the proteases are alkaline or neutral proteases. Method according to claim 1 or 2, wherein the pH is between 4.2 and 4.8. Process according to claims 1 to 3, wherein the pH is between 4.5 and 4.7. Process according to claims 1 to 4, wherein proteases are used which are at a pH above 6 are active. Process according to claims 1 to 5, wherein the enzymes are inactivated between step b) and c). Method according to claim 6, wherein the enzymes are inactivated by heat. Process according to claims 1 to 7, wherein the gluten is wheat gluten. Peptide preparation made from gluten protein is glutamine-rich and gluten-free and by a method according to claims 1 to 8 available is. A peptide preparation according to claim 9, wherein the gluten, from which the preparation is made is wheat gluten. A peptide preparation according to claims 9 and 10 for use as an ingredient in glutamine peptide tablets. A peptide preparation according to claims 9 and 10 for use as an ingredient in glutamine peptide liquid drinks. A peptide preparation according to claims 9 and 10 for use as an ingredient in glutamine-peptide-natural food. Glutamine peptide tablets, which are the usual Carrier, thinner and excipients for tablets and a peptide preparation according to claims 9 and 10 as a glutamine peptide source. Glutamine peptide liquid drink, the the usual Ingredients for beverages and a peptide preparation according to claims 9 and 10 as a glutamine peptide source. Glutamine peptide basic nutrition, which is the usual Carrier, thinner and excipients for enteral nutrition and a peptide preparation according to claims 9 and 10 as a glutamine peptide source.